Prakash Group Page

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2003 Groups

N*Synch

Olmec

Young Power

Prakash

Biobricks

 

Design Goal Methods References
               

Applications Link; This link will go to a  common page for all groups describing why we did these projects. (Do not work on this for each group, we'll take care of it.

Background
For more information about Synthetic Biology visit the Synthetic Biology web pages at MIT.

For more information about Biobricks visit the Biobricks page.

For more information about the repressilator system see this article. 

Design Goal

Synchronization of a repressilator:

The above scheme uses bacterial cell-cell signalling to synchronise repressilators among neighbouring bacteria (E.coli). The repressilator is based on the Elowitz repressilator but is not identcal in terms of the order of the repressors. In other words, if the Elowitz oscillator is counter-clockwise, our design is clockwise. The Vibrio fischeri LuxI-LuxR quorum sensing circuit components were particularly suited because of their simplicity and also as they have been shown to work in E.coli at the temperature range the repressilator functions in. The magenta squiggles represent the possible use of one of two alternative promoters for the tetR and luxI genes. This leads to four alternative designs each with its own pros and cons. Simulations carried out by our group did not help us in choosing any one particular design over another. So we have proposed a strategy to synthesise the four combinations.

Description of System

Theorized Biological Mechanism

 

 

Graphical Representation

Parts List

We intend to assemble our parts in modules as shown in the table. During assembly, we will store every completed module as a separate biobrick to facilitate debugging. Proposed debug strategies for each part are included in the last column.

To help synthesise the above four designs without duplication of effort, all the parts coloured blue in the table (common to all designs) can be connected into blocks first and which can then be connected to each other with the appropriate red colored parts.

The designs have to be assembled in sequence from top to bottom and circularised to yield a viable plasmid.

MODULE Part Type DESIGN 1 DESIGN 2 DESIGN 3 DESIGN 4 NOTES (including debug strategies)
             
Backbone            
  BACKBONE plasmid BBa_B0001  BBa_B0001 BBa_B0001 BBa_B0001 Try plasmids with different copy numbers and choose best
             
Oscillator Core            
 

cI-LuxR hybrid (Design 1,2)

cI repressible (Design3,4)

BBa_R0065 BBa_R0065 BBa_R0051 BBa_R0051 Choice of making "oscillator core" dependent on synchronisation circuit is at the risk of increasing the period but it could make the design more robust
  RBS BBa_B0032 BBa_B0032 BBa_B0032 BBa_B0032 Weak RBS preferred
  tetR-LVA BBa_C0040 BBa_C0040 BBa_C0040 BBa_C0040 Same degradation tag used on all oscillating components
  Terminator1 BBa_B0010 BBa_B0010 BBa_B0010 BBa_B0010  
  Terminator2 BBa_B0012 BBa_B0012 BBa_B0012 BBa_B0012  
  tetR repressible BBa_R0040 BBa_R0040 BBa_R0040 BBa_R0040  
  RBS BBa_B0032 BBa_B0032 BBa_B0032 BBa_B0032 Weak RBS preferred
  lacI-LVA BBa_C0012 BBa_C0012 BBa_C0012 BBa_C0012 Same degradation tag used on all oscillating components
  Terminator1 BBa_B0010 BBa_B0010 BBa_B0010 BBa_B0010  
  Terminator2 BBa_B0012 BBa_B0012 BBa_B0012 BBa_B0012  
  lacI repressible BBa_R0010 BBa_R0010 BBa_R0010 BBa_R0010  
  RBS BBa_B0032 BBa_B0032 BBa_B0032 BBa_B0032 Weak RBS preferred
  cI-LVA BBa_C0051 BBa_C0051 BBa_C0051 BBa_C0051 Same degradation tag used on all oscillating components
  Terminator1 BBa_B0010 BBa_B0010 BBa_B0010 BBa_B0010  
  Terminator2 BBa_B0012 BBa_B0012 BBa_B0012 BBa_B0012  
             
Synchronising Circuit            
  constitutive BBa_R0063 BBa_R0063 BBa_R0063 BBa_R0063  
  RBS BBa_B0030 BBa_B0030 BBa_B0030 BBa_B0030 Strong RBS preferred
  LuxR w/o LVA BBa_C0062 BBa_C0062 BBa_C0062 BBa_C0062 Note the absence of LVA.LuxR needs to be present at high and steady levels so that weak HSL signals from neighbouring cells can be picked up.
  Terminator1 BBa_B0010 BBa_B0010 BBa_B0010 BBa_B0010  
  Terminator2 BBa_B0012 BBa_B0012 BBa_B0012 BBa_B0012  
 

cI-LuxR hybrid (Design 1,3)

cI repressible (Design 2,4)

BBa_R0065 BBa_R0051 BBa_R0065 BBa_R0051 BBaR0065 will amplify the signal as in the natural system and give rise to potentially stronger synchronisation
  RBS BBa_B0030 BBa_B0030 BBa_B0030 BBa_B0030 Try different RBS
  LuxI-LVA BBa_C0061 BBa_C0061 BBa_C0061 BBa_C0061 May need two or more copies of LuxI LVA gene if the rate of HSL synthesis is too slow. This can be achieved using polycistronic design without upsetting the stoichiometry of repressor binding.
  Terminator1 BBa_B0010 BBa_B0010 BBa_B0010 BBa_B0010  
  Terminator2 BBa_B0012 BBa_B0012 BBa_B0012 BBa_B0012  
  lacI repressible BBa_R0010 BBa_R0010 BBa_R0010 BBa_R0010

Simulations indicate BBa_R0010 is a better choice than BBa_R0040 but this could also be tried.

  RBS BBa_B0030 BBa_B0030 BBa_B0030 BBa_B0030 Try different RBS
  aiiA-LVA BBa_C0060 BBa_C0060 BBa_C0060 BBa_C0060 This enzyme has zinc in its active site and may need the addition of Zn to the medium for optimal function. It degrades the auto-inducer HSL. The system requires that there is an efficient degradation mechanism for HSL. Additional mechanisms such as flow of medium may be needed.
  Terminator1 BBa_B0010 BBa_B0010 BBa_B0010 BBa_B0010  
  Terminator2 BBa_B0012 BBa_B0012 BBa_B0012 BBa_B0012  
             
Reporter 1           This could be in a separate plasmid or a separate strain.
  tetR repressible BBa_R0040 BBa_R0040 BBa_R0040 BBa_R0040  
  RBS BBa_B0030 BBa_B0030 BBa_B0030 BBa_B0030  
  CFP-LVA BBa_E0022 BBa_E0022 BBa_E0022 BBa_E0022 Same degradation tag used on all oscillating components
  Terminator1 BBa_B0010 BBa_B0010 BBa_B0010 BBa_B0010  
  Terminator2 BBa_B0012 BBa_B0012 BBa_B0012 BBa_B0012  
             
Reporter 2           This could be in a separate plasmid or a separate strain.
  cI repressible BBa_R0040 BBa_R0040 BBa_R0040 BBa_R0040  
  RBS BBa_B0030 BBa_B0030 BBa_B0030 BBa_B0030  
  YFP-LVA BBa_E0031 BBa_E0031 BBa_E0031 BBa_E0031 Same degradation tag used on all oscillating components
  Terminator1 BBa_B0010 BBa_B0010 BBa_B0010 BBa_B0010  
  Terminator2 BBa_B0012 BBa_B0012 BBa_B0012 BBa_B0012  
             
             
             

Methods

Simulations

Summary

Call the wrapper files to run the simulations. The colony simulation is connected in a star topology, such that the only variable coupling the individual cells is the environmental HSL concentration.

Sample Graphs

Source Codes

Single cell: simplest_repressilator.m (2k), simplest_wrapper.m (1k)
Colony with uniform environmental HSL concentration: HSL.m (4k), HSL_wrapper.m (3k)

Challenges and Debug Plan

Even before assembly: Do all the parts work?

It may be worthwhile to test individual RBS, promoters, repressors and reportes before assembling the parts as most of them have been modified to meet the biobricks format and also because it is possible that we do not know enough about their biology. A standard reporter can be chosen to measure the function of all the parts so their their relative strengths can be compared.

Testing the repressilator independent of the synchronisation circuit

The Elowitz repressilator design has not been tested for different configurations of repressors. We suggest that our design of the synchrollator be tested independently of the quorum sensing synchronising circuit for its repressilator-like behaviour(the design is modular to facilitate this). At the next stage, various synchronising strategies could be tried out. Tuning the RBS parts are a good way to "tune" the repressilator till it works in the current configuration.

HSL (the autoinducer) is commercially available in pure form and will prove useful in debugging the synchronisation module.

 

Testing Intercellular Communication

In order to test whether the homoserine-lactone (HSL) autoinducer (AI) diffuses between the cells, one can make a HSL-dependent reporter cell. In the scheme below, HSL is produced in the synchronized repressilator. The reporter cell contains all the lux genes except luxI, which is responsible for HSL production. In the lux system, bioluminescence is promoted by the activated luxR-AI complex. However, since the reporter cell lacks the ability to produce the HSL autoinducer, bioluminescence by the reporter cell is evidence that the HSL is indeed diffusing between cells.

In order that the coculture of the two cell strains (reporter strain and the repressilator strain) be sustained, we could engineer them each degrades one of two antibiotics and culture them in presence of both antibiotics. Both strains would be needed to deplete the antibiotics from the medium. Other strategies such as each strain makes one of two essential amino acids that the other cannot make could also be tried.

Degradation of acyl-homoserine lactone

The half life of HSL is 24 hrs at pH of 7.5 and it is important that a degradation mechanism that is faster than the desired period of oscillations be introduced into the system. Cyclical degration of HSL would generate better synchronisation signals than a constant degradation mechanism. Possible degradation mechanisms that were considered were:

1. Flow of medium through the filter that washes away the HSL secreted into the medium at a constant rate.

2. aiiA is an enzyme that inactivates acyl-HSL, presumably by cleaving the acyl side-chain. aiiA could be placed under the control of a tetR repressible promoter or a lacI repressible promoter.

Placing aiiA under the control of lacI repressible promoter seemed to be the best design as per our simulations. However, it may be worthwhile to try other possibilities.

It may be necessary to test if the LVA-tagged aiiA enzyme is functional. This can be done by introducing both the LuxR-LuxICDABE bioluminescence cassette and the aiiA gene in E. coli and measuring the density at which bioluminescence is observed.

 

List of known inhibitors of the system components

The following compounds can be used to debug the system by perturbing one component of the system at a time.

anhydrotetracycline - inhibits tetR repressor

IPTG - inhibits lacI repressor

halogenated furanones - degrade LuxR activator. These compounds are synthesised by a marine alga Delisea pulchra and the genes that synthesise them are not known yet. They can be isolated from Delisea pulchra. They bind LuxR and degrade it in a protease independent manner.

 

Future Research

Comments for future projects groups

References

Quorum sensing in bacteria. Miller MB, Bassler BL. Annu Rev Microbiol 2001;55:165-99

Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology 2002 Apr;148(Pt 4):1119-27

A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000

Octamerization of lambda CI repressor is needed for effective repression of P(RM) and efficient switching from lysogeny.Genes Dev 2001 Nov 15;15(22):3013-22

Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species.Dong YH, Gusti AR, Zhang Q, Xu JL, Zhang LH. Appl Environ Microbiol 2002 Apr;68(4):1754-9

 

Group Members

Vinay S Mahajan

Voichita D. Marinescu

Brian Chow

Alexander D. Wissner-Gross

Peter Carr